Macrobrachium nipponense, contains many neurosecretory hormones affecting the process of reproduction, molting, metabolism of glucose, and other functions. In this study, important metabolic pathways and candidate genes involved in male sexual development were selected from M. nipponense. The methodology involved performing long-read and next generation transcriptome sequencing of genes from the androgenic gland after eyestalk ablation. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation of both the single-side (SS) and double-side (DS) eyestalk, compared with the control group (CG). The long-read transcriptome generated 49,840 non-redundant transcripts. A total of 1319, 2092 and 4351 differentially expressed genes (DEGs) were identified between CG versus SS, SS versus DS and CG versus DS, respectively. These data indicated that ablation of the double-sided eyestalk played stronger regulatory roles than the single-side ablation on male sexual development in M. nipponense. This was consistent with the qPCR analysis. Cell Cycle, Cellular Senescence, Oxidative Phosphorylation, Glycolysis/Gluconeogenesis and Steroid Hormone Biosynthesis were the primary enriched metabolic pathways in all three comparisons, and the important genes from these metabolic pathways were also selected. qPCR permitted secondary confirmation of ten DEGs identified through RNA-seq. RNAi-mediated silencing analyses of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has a positive regulatory effect on testes development. This study provides valuable insight into male sexual development in M. nipponense, including metabolic pathways and genes, paving the way for advanced studies on male sexual development in this species and in other crustaceans.The eyestalk of crustaceans, such as  Macrobrachium nipponense , is widely distributed in China and other Asian countries3. This species is commercially important with an annual aquaculture production of ~ 205,010 tons in 20164. Similar to other Macrobrachium species, male prawns grow faster and reach a larger size at harvest time2. Thus, male prawns are preferred in the M. nipponense aquaculture industry. In addition, the rapid development of testes in the reproductive season is another issue restricting the sustainable development of M. nipponense. Previous studies revealed that the testes of M. nipponense reach sexual maturity within 40 days after hatching5. Thus, inbreeding will occur between the newborn prawns. Inbreeding will lead to a decrease in resistance to adversity in their offspring, small scale of market prawn, and a degradation in germplasm resources. Therefore, it is important to fully understand the mechanism of male sexual differentiation and development with the aim of producing all-male progeny on a commercial scale. This can be accomplished by regulating the process of testes development in M. nipponense.The oriental river prawn, 7. For example, the ablation of the androgenic gland from male M. rosenbergii resulted in the sex reversal; a phenomenon that has been termed “neo-female”7. The insulin-like androgenic gland hormone (IAG) is an important hormone secreted by the androgenic gland. IAG was shown to promote male sexual differentiation and development in many crustacean species10. It is the most important male sex-related gene acknowledged in many crustacean species. Knockdown of IAG expression by RNAi in male M. rosenbergii can also result in sex reversal11. IAG, specially expressed in the androgenic gland of M. nipponense and other crustaceans, was also implicated in the maintenance of male secondary sex characteristics, spermatogenesis, reproductive strategies and the regulation of growth15. Recent studies on the androgenic gland have become more prominent in the literature. A series of transcriptomes from the androgenic gland have been constructed in M. nipponense18, and a series of important genes from the androgenic gland have been implicated as having an essential role in male sexual development22. In addition, the histological observations during different post-larval developmental stages indicated that the development of the androgenic gland has regulatory roles in the development of testes5.The androgenic gland is a unique tissue in crustacean species. It has been shown to play an essential role in male sexual differentiation and development in crustacean species. Many studies have reported that the androgenic gland and its secreted hormones promote the driving of male sexual differentiation, the establishment of male sexual characteristics and the development of the testes in crustacean species23. It stores and releases the crustacean hyperglycemic hormone (CHH) superfamily of neurohormones, including CHH, ion transport peptides (ITP), gonad-inhibiting hormone (GIH), molt inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH), playing essential roles in reproduction26, molting29, metabolism of glucose31 and other function34. Knockdown of GIH by RNAi promoted ovarian development in M. nipponense35. Knockdown of MIH by RNAi promoted molting in M. nipponense36. CHH has been shown to promote testes development in M. nipponense37.The eyestalk of crustacean species has many neurosecretory hormones. The X-organ–SG complex (XO–SG) was identified as a principal neuroendocrine gland located in the eyestalkM. nipponense were selected by performing long-read and next generation transcriptome profiling analysis of the androgenic gland after the ablation of the single-side and double-side eyestalk. The functions of Hydroxysteroid dehydrogenase like 1 (HSDL1), which was predicted to be involved in the mechanism of male sexual development in this study and in a previous study38, were further analyzed in depth by using qPCR analysis and RNAi. This study provides valuable evidence of male sexual differentiation and development in M. nipponense, as well as in other crustacean species.In this study, the vital metabolic pathways and genes involved in male sexual differentiation and development in Mn-IAG was measured in three groups: control group (CG), single-side ablation of eyestalk (SS), and double-side ablation of eyestalk (DS) . However, Mn-IAG expression was only slightly higher at day 7 than day 4 in both the SS and DS groups, showing no significant difference (P > 0.05). Mn-IAG expression in the DS group was ~ 2-folder higher than that in the SS group on the same day and showed significant difference (P < 0.05).The mRNA expression of DS) Fig. . The datM. nipponense genome, and a total of 43,115 unigenes matched known sequences in the M. nipponense genome. All of the non-redundant transcripts were then compared with the Nr database (non-redundant protein database) and nucleotide sequences in NCBI in order to identify their putative functions , M. nipponense  and Stegodyphus mimosarun . The unigenes were further matched with the known genomes of crustacean species, including Litopenaeus vannamei, Eriocheir sinensis, Portunus trituberculatus, and Drosophila melanogaster. A total of 3067, 3263, 3198 and 167 unigenes matched the known proteins with the genome of L. vannamei, E. sinensis, P. trituberculatus, and drosophila fly, respectively. Generally speaking, the unigenes of M. nipponense transcriptome showed the highest sequence identities with that of E. sinensis.A total of 22.83 GBs of clean data were generated in the long-read transcriptome. A total of 160,496 high-quality transcripts were obtained with a mean length of 2230 bp. Finally, 49,840 non-redundant transcripts were identified in the long-read transcriptome. These unigenes were annotated, based on the ns Table . A totalwww.kegg.jp/kegg/kegg1.html). A total of 18,618 (36.72%) unigenes were highly matched known genes in the KEGG database, mapped onto 264 metabolic pathways.Gene Ontology (GO) and Cluster of Orthologous Groups (COG)analysis aimed to provide a structured vocabulary to describe gene products. A total of 19,673 (39.76%) unigenes were assigned to the GO database comprised of 52 functional groups  were identified, using the criterion of > 2.0 as up-regulatory genes and < 0.5 as down-regulatory genes, and with a MAD2A, Polo-like kinase 1, Cyclin A, cyclin-dependent kinase 2 (Cdk2) and Cyclin B were found in the metabolic pathways of Cell cycle and Cellular senescence, which were differentially expressed in all three comparisons. Succinate dehydrogenase complex iron sulfur subunit B Gene (SDHB), Cytochrome c oxidase assembly protein COX11 and Cytochrome c oxidase subunit 7A1 were selected from the metabolic pathways of Oxidative Phosphorylation. Acetyl-coenzyme A synthetase 2-like, Fructose-bisphosphate aldolase and Alcohol dehydrogenase class-3 were differentially expressed in the metabolic pathways of Glycolysis/Gluconeogenesis. Estrogen Sulfotransferase, 3 beta-hydroxysteroid dehydrogenase and HSDL1 were identified from the metabolic pathways of Steroid Hormone Biosynthesis.A total of 15 DEGs were selected from these enriched metabolic pathways, which are listed in Table MAD2A . The mRNA expression of estrogen sulfotransferase  , whereas a significant difference was observed in DS prawns (P < 0.05). The mRNA expression of HSDL1 , whereas a significant difference was found in CG prawns (P < 0.05).qPCR analysis was used to verify the expressions of important DEGs in the androgenic gland from the CG, SS, and DS prawns. We selected 10 out of 15 DEGs to verify the accuracy of RNA-seq. The qPCR analysis showed the same expression pattern as the RNA-seq Fig. . Six DEGD2A Fig. B, Polo-lD2A Fig. C, CyclinD2A Fig. D, Cdc2 kD2A Fig. E and CycD2A Fig. F. The mRase Fig. G and alc-3) Fig. H showed DL1 Fig. I and SDHDHB Fig. J betweenMn-HSDL1 mRNA showed the highest expression level in the hepatopancreas, followed by the testis, which showed significant difference with other tested tissues (P < 0.05)38. The mRNA expression of Mn-HSDL1 in different prawn developmental stages was measured by using qPCR , while it showed no significant difference with other tested stages (P > 0.05). During the post-larval developmental stage, the lowest expression level was observed in post-larval day 5 (PL5), and then gradually increased. The highest expression level was observed in PL25♂, which was 3.72 and 1.94-folder higher than PL5 and PL25♀, respectively.Previous studies have shown that s P < 0.038. The mMn-HSDL1 on male sexual development in M. nipponense. qPCR analysis revealed that the expression of Mn-HSDL1 remained stable in the control group after the injection of Mn-HSDL1 dsRNA (P > 0.05). However, the expression of Mn-HSDL1 significantly decreased by 96% and 90% at day 7 and 14, respectively, following the injection of Mn-HSDL1 dsRNA as compared with the control group . In the RNAi group, the expression of Mn-IAG was significantly decreased at day 7 and day 14 after the injection of Mn-HSDL1 dsRNA. Specifically, the expression decreased by ~ 61% and 54% at day 7 and 14, respectively, compared with the control group (P < 0.05).The expression of wns Fig. B. AccordMn-HSDL1 dsRNA treatment. Sperm were rarely found at day 14 after Mn-HSDL1 dsRNA treatment. The percentages of sperm decreased from 57.69% at Day 1 to 1.27% at Day 14 in RNAi group , Insulin-like growth factor 2 (IGF2), Cytochrome P450 (CYP11) and 5′-AMP-activated protein kinase catalytic subunit alpha-2 (PRKAA2) in the previous studies40. The regulatory effects of Mn-HSDL1 with Mn-IGF1, Mn-IGF2, Mn-CYP11 and Mn-PRKAA2 were measured in the same cDNA template of RNAi by using qPCR. According to the qPCR analysis, the expressions of Mn-CYP11 and Mn-PRKAA2 were decreased with the decrease of Mn-HSDL1, which showed positive regulatory effects , functioning in both S-phase and mitosis51. Cdk1/cyclin B, also known as maturation promoting factor (MPF), is one of the main protein kinases. It activates, and serves as master regulator, for the M-phase transition, phosphorylating and activating other downstream protein kinases, and directly phosphorylating several structural proteins involved in cellular reorganization54. The Cdk family includes eight Cdk genes that can combine with different types of cyclins to form complexes, regulating the process of cell transition from the G1 phase to the S phase or G2 phase to the M phase and finally exiting from M phase. Cdk2 in particular is a member of a highly conserved family of protein kinases, regulating the eukaryotic cell cycle57.The transcriptome profiling analysis revealed that Cell Cycle and Cell Senescence are the most enriched metabolic pathways in all three comparisons. The Cell Cycle is a ubiquitous and complex process that ensures correct cell proliferation. This pathway is crucial for the prevention and/or correction of damaged DNA, genetic abnormalities and mutations, with cyclins and cyclin-dependent kinases functioning in this process58. Glycolysis/Gluconeogenesis promotes the conversion of glucose (C6H12O6) into pyruvate (CH3COCOO−  + H+), releasing free energy to form ATP and reduced nicotinamide adenine dinucleotide59. Three DEGs were selected from Oxidative Phosphorylation and Glycolysis/Gluconeogenesis. SDHB, a DEG that was down-regulated between CG versus SS and CG versus DS. SDHB, was also predicted to be involved in the mechanism of male sexual development in M. nipponense38. SDHB is one of four protein subunits that form succinate dehydrogenase, which catalyzes the oxidation of succinate61. Two subunits of cytochrome c oxidase, which function during oxidative phosphorylation, were also differentially expressed. These two subunits included cytochrome c oxidase assembly protein COX11 and cytochrome c oxidase subunit 7A1. Cytochrome c oxidase is located at the end of the cytochrome c system in cellular respiration. This enzyme directly transfers the electrons of respiratory substrates to molecular oxygen through the cytochrome system63.Adenosine-triphosphate (ATP), a high-energy compound used as an energy source in nearly all metabolic activities, is essential for male differentiation and development. Therefore, it is of interest that in the present study, Oxidative Phosphorylation and Glycolysis/Gluconeogenesis were the main enriched metabolic pathways in all three comparisons. Oxidative Phosphorylation occurs in the inner membrane of mitochondria of eukaryotic cells or in the cytoplasm of prokaryotes. The energy released from the oxidation of substances in vivo promotes the coupling reaction between adenosine diphosphate (ADP) and inorganic phosphate to synthesize ATP through the respiratory chain65. Hormones are generally divided into five main classes: glucocorticoids, mineralocorticoids, androgens, estrogens, and progestogens. Natural steroid hormones, which are lipids, are generally synthesized from cholesterol in the gonads and adrenal glands67. HSDL1 was differentially expressed between CG versus SS and CG versus DS, indicating that the expressions of HSDL1 is significantly regulated by the ablation of both single-side eyestalk and double-side eyestalk. HSDL1 was also shown to be involved in the mechanism of male sexual development in a previous study38. The short-chain dehydrogenase/reductases family (SDR) is a large enzyme family, which can affect mammalian reproduction, hypertension, neoplasia, and digestion69. Hydroxysteroid dehydrogenase is a subfamily of SDR, that functions in sex-determination, establishment and maintenance of secondary sexual characteristics, and the regulation of the endocrine system through catalyzing the metabolism of steroid hormones. HSDL1 is an important gene in the metabolic pathway of steroid hormones70. qPCR verification revealed that the expression pattern of important DEGs from these metabolic pathways were the same as the RNA-seq data  in humans, as revealed by Northern Blot analysis70. In situ hybridization indicated that the expression of HSDL1 was higher in prostate cancer than that in normal prostate tissue. In addition, this gene is involved in the fetal sheep development in the late gestational stages71. The qPCR analysis in different mature tissues revealed that the highest expression level of Mn-HSDL1 was observed in hepatopancreas, followed by testes, while Mn-HSDL1 RNA was rarely detected in other tissues38. Thus, it is predicted that HSDL1 may be involved in testes development in M. nipponense. RNAi was further used to analyze the potential functions of Mn-HSDL1 in testes development. The mRNA expression of Mn-HSDL1 was significantly decreased at day 7 and day 14 after Mn-HSDL1 dsRNA injection . It received considerable attention because of the activation of both glucose and lipid metabolism and improvement of the insulin sensitivity76. However, Mn-HSDL1 negatively affects expression of Mn-IGF1 and Mn-IGF2. A reasonable explanation for this is that silencing Mn-HSDL1 inhibited testis development. Thus, the energy for testis development was used to promote the growth performance78.Previous studies reported that HSDL1 has a regulatory relationship with M. nipponense. qPCR analysis revealed that Mn-IAG expression significantly increased after the ablation of both single-side and double-side eyestalks, indicating that the ablation of eyestalk has potential regulatory roles on the process of male sexual development in M. nipponense. The long-read transcriptome generated 49,480 non-redundant transcripts. A total of 1319, 2092 and 4351 DEGs were identified between CG versus SS, SS versus DS and CG versus DS, respectively, indicating that the ablation of double-side eyestalks plays a regulatory role on male sexual development. Cell Cycle, Cellular Senescence, Oxidative Phosphorylation, Glycolysis/Gluconeogenesis and Steroid Hormone Biosynthesis were the main enriched metabolic pathways in all three comparisons, and the important DEGs from these metabolic pathways were identified. qPCR analysis and RNAi analysis of Mn-HSDL1 indicated that HSDL1 has positive regulatory effects on testes development. Overall, this study provided valuable information concerning the mechanisms underlying male sexual development in M. nipponense and potentially other crustacean species as well.In conclusion, candidate genes that are possibly involved in the male sexual development were selected through performing long-read and next generation transcriptome sequencing of the androgenic gland after eyestalk ablation in Permission was obtained from the Tai Lake Fishery Management Council and the committee of Freshwater Fisheries Research Center during the experimental programs. MS222 anesthesia was used to sedate the prawns and shear the tissues. All experiments were performed in accordance with relevant guidelines and regulations. Authors complied with the ARRIVE guidelines.M. nipponense were collected from a wild population in Tai Lake in July, Wuxi, China . The body weight of male prawns was 3.63–4.94 g and the body weight for females was 3.21–3.45 g. All samples were randomly divided and transferred to three, 500 L tanks and maintained in aerated freshwater for three days. The three groups in this study were: CG, SS, and DS. The androgenic glands were collected from the three groups after 7 days of eyestalk ablation, and immediately preserved in liquid nitrogen until used for long-read and next-generation transcriptomic analysis. Mature tissues that were studied included testes ovaries, hepatopancreas, muscle, eyestalk, gill, heart and brain. One male parent prawn with a body weight of 4.87 g and one female parent prawn with a body weight of 3.45 g were collected from the wild population and mated in the laboratory in order to produce the full-sibs population. Specimens for the different stages of larval and post-larval developmental stages were obtained from the full-sibs population after hatching and collected throughout the maturation process.A total of 600 healthy male prawns and 20 healthy female prawns of 79.In order to provide sufficient RNA with an aim to establish a reference transcriptome for further analysis, equal amount of androgenic gland tissue from the CG, SS, and DS groups (N ≥ 60) were pooled together to perform the long-read sequencing. According to the manufacturer’s instructions, the UNlQ-10 Column Trizol Total RNA Isolation Kit  was used to extract total RNA, and an Agilent RNA 6000 Nano kit and chips on a Bioanalyzer 2100  was used to measure the RNA integrity. A PacBio RSII platform  was employed to construct the long-read transcriptome. The detailed procedures for the construction of long-read transcriptome and the analysis of raw sequence data have been well described in our previous study80, and the LRS isoforms were annotated81. Using Blastp, the transcriptome factors were aligned to the PlnTFDB database (http://plntfdb.bio.uni-potsdam.de/v3.0/), the AnimalTFDB database , and the CARD database (https://card.mcmaster.ca/) for the selection of genes involved in the mechanism of male sexual development in M. nipponense, using the threshold of E-value ≥ 1e–10. Finally, all Blastp results were processed with BLAST2GO82 for functional annotation. The long-read were annotated in the M. nipponense genome by using Lorean83.In the next step, the contaminant sequences were removed by stepwise CLC42.The comparative transcriptome analysis of the androgenic gland between the CG, SS and DS groups were performed. In order to ensure the sufficient amount of RNA samples, androgenic glands from at least 30 prawns were pooled to form one biological replicate, and three biological replicates were sequenced for all three groups. Previously published studies have described the experimental process84. The NR protein, the GO, the COG and the KEGG database were then used to perform the gene annotation, using an E-value cut-off of 10−516. Blast2go software was used for functional annotation by GO terms82. Blast software was employed to perform the functional annotation against the COG85 and KEGG86 database. EB-seq algorithm was used to filter the differentially expressed genes, under the criteria of FDR  < 0.0587.Clean reads were assembled into non-redundant transcripts by using the Trinity program (version: trinityrnaseq_r20131110)Mn-HSDL1 in different developmental stages, as well as for confirmation of DEGs. The Bio-Rad iCycler iQ5 Real-Time PCR System (Bio-Rad) was used to carry out the SYBR Green RT-qPCR assay. The procedure has been well described in previous studies22. The primers used for qPCR verification of important DEGs are listed in Table Mn-HSDL1 are listed in Table 88. Three replicates were performed for each tissue.qPCR was used to measure the relative mRNA expression of Mn- HSDL1 in male sexual development in M. nipponense. The Snap Dragon tool was used to design the specific RNAi primer with the T7 promoter site (http://www.flyrnai.org/cgibin/RNAifind_primers.pl) shown in Table Mn-HSDL1 dsRNA, according to manufacturer’s instructions. A total of 300 healthy mature male M. nipponense with a body weight of 3.21–4.78 g were collected and divided into two groups. As described in the previous study90, prawns from the experimental group were injected with 4 μg/g Mn- HSDL1 dsRNA, while prawns from the control group were injected with an equal volume of GFP dsRNA (control). HSDL1 mRNA expression was investigated in the androgenic gland by qPCR 1, 7 and 14 days after the injection, permitting confirmation of silencing efficiency (N ≥ 5). mRNA expression of Mn-IAG was measured in the same cDNA templates in order to analyze the regulatory relationship between Mn-HSDL1 and Mn-IAG.RNAi was performed to analyze the potential regulatory roles of 92. Olympus SZX16 microscope was used to observe the slides . The various cell types were labeled based on morphological analysis5.The morphological changes in the testes between different days after RNAi treatment were observed by Hematoxylin and eosin (HE) staining. Five testicular samples were collected after 1, 7, and 14 days of RNAi treatment for HE staining. The procedures have been well described in previous studiesP < 0.05).Quantitative data were expressed as mean ± SD. Statistical differences were estimated by one-way ANOVA followed by LSD and Duncan’s multiple range test. All statistics were measured using SPSS Statistics 23.0. A probability level of 0.05 was used to indicate significance (Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Figure 5.Supplementary Figure 6.Supplementary Table S1.


Objective: To study whether standard cognitive behavioral therapy (CBT) and a shorter, interpersonal oriented cognitive behavioral therapy (I-CBT) can improve physical function and fatigue in patients diagnosed with mild to moderate chronic fatigue syndrome (CFS) in a multidisciplinary fatigue clinic.Design: Consecutively 236 participants 18–62 years old meeting the Centre of Decease Control, CDC 1994 criteria, with a subsample also fulfilling the Canadian criteria for CFS, were randomly allocated to one of three groups. Two intervention groups received either 16 weeks of standard CBT or 8 weeks of I-CBT vs. a waiting-list control group (WLC). Primary outcome was the subscale Physical Function (PF) from SF-36 (0–100). Secondary outcome was amongst others fatigue measured by Chalder Fatigue Questionnaire (CFQ) (0–33). Outcomes were repeatedly measured up to 52 weeks from baseline.Results: The additional effect relative to baseline at post-intervention for SF-36 physical function was 14.2 (95% CI 7.9–20.4 p < 0.001) points higher for standard CBT and 6.8 (0.5–13.2 p = 0.036) points higher for I-CBT compared with the control group. The additional effect relative to baseline at post-intervention for fatigue was 5.9 (95% CI 0.5–10.5 p = 0.03) points lower for standard CBT compared with the control group but did not differ substantially for I-CBT 4.8 (95% CI −0.4 to 9.9 p = 0.07). The positive change in physical function persisted at 1-year follow-up for both treatment groups, and for standard CBT also in fatigue. The two intervention groups did not differ significantly in self-reported physical function and fatigue at the 1-year follow-up. No serious adverse reactions were recorded in any of the groups during the trial period.Interpretation: A 16-week standard, individual CBT intervention improves physical function and fatigue in CFS outpatients with mild to moderate disease. A shorter 8-week I-CBT program improves physical function. Both treatments are safe, and the effect persist 1 year after baseline.Clinical Trial registration:ClinicalTrials.gov, Identifier: NCT00920777, registered June 15, 2009.REK-project number: 4.2008.2586, registered April 2, 2008. Funding: The Liaison Committee for Education, Research and Innovation in Central Norway. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), is an illness characterized by unexplained, severe fatigue and post exertional malaise with additional symptoms including, cognitive impairment, sleep disturbance, sensory hypersensitivity, headache, pain in muscles and joints, irritable bowel and intermittent flu-like symptoms –4. The iThe terms CFS and ME are often used interchangeably and in combination, CFS/ME, ME/CFS. Until recently, there has been no international consensus on whether ME and CFS are separate conditions or similar enough to belong under an umbrella term such as CFS/ME . In thisDue to terminological variations and diagnostic inconsistencies, it is difficult to assess the prevalence and incidence rate of CFS in a population. Overall prevalence estimates worldwide vary from 0.1 to 2.5%, depending on the diagnostic criteria used , 13.The etiology of CFS is largely unknown. Nevertheless, several possible predictors and associative mechanisms have been hypothesized, including various infections, prolonged strain and physical trauma , 15, an individual cognitive behavior therapy (CBT) and graded exercise therapy (GET) have until now been the best-documented treatments for mild to moderate CFS, also in combination, but the effect has been found to be moderate  vs. a waiting list control group (WLC) and assess the possible treatment effect sustainability. Systematic reviews on treatment and management of chronic fatigue syndrome have found little evidence concerning the appropriate duration of follow-up of interventions used in the management of CFS , 32. HowThis study was a consecutive randomized controlled open-label trial with outcome assessed up to 52 weeks after baseline for patients meeting the CDC 1994 criteria  for CFS.The trial was registered and approved by the Norwegian Regional Committee for Medical and Health Research Ethics  April 2, 2008, before inclusion of patients. There was a delay in registration of the trial in Clinical Trials , because the research group was not aware of the registration requirements at the time. The authors confirm that all ongoing and related trials for this intervention are registered. In all, 626 patients with unspecified fatigue referred from their GP were assessed for eligibility at the CFS/ME clinic at St. Olav‘s Hospital in Trondheim between October 6, 2008, and September 12, 2012 . Of the All referrals to the clinic were screened by an experienced multidisciplinary team consisting of a doctor , a clinical psychologist or psychiatrist, a physical therapist, and a registered nurse. Blood samples were taken from each patient in accordance with the Norwegian Directorate of Health's requirement for assessing patients with suspected CFS , includiThe physical therapist tested the patient's physical condition, including an indirect VO2max test, muscle tonus, tender points and a brief neurological examination. The psychologist or psychiatrist used a structured interview M.I.N.I   to assesAfter baseline assessment and having obtained a written consent, participants were allocated to either one of the two treatment groups or to the control group by computer-generated sequence, provided by the Section for Applied Clinical Research (ACR) at NTNU. Once notified of treatment allocation by the ACR, the research coordinator informed the participant, first by phone and later in a letter including practical information and a treatment plan. The participants started treatment within 3 weeks after randomization. Participants and therapists could not be masked to treatment allocation. Both therapist and participant knew the length and contents of the therapy that was given. The participants scored the primary and secondary outcomes in questionnaires. The statistician was masked to treatment groups when analyzing the outcome data, but the difference in number of measuring points made total masking a challenge.Cognitive behavioral therapy (CBT) is a form of psychotherapy with assumed moderate effect for many patients with mild to moderate CFS . StandarThe 16-week intervention of individual, standard CBT was given by three trained cognitive therapists at St. Olav's Hospital, Trondheim, Norway. The actual therapy manual was based on previous manuals from similar trials , 45, andThe 8 weeks of individual, interpersonal and personality-oriented CBT was given by four trained cognitive therapists at the private health centre Coperio, Trondheim, Norway. The treatment manual was developed by co-author (TCS). The intervention consisted of 8 weekly therapy sessions and a booster session 4 weeks after the eighth session. I-CBT is a new form of treatment for CFS/ME. Its treatment effects have thus not been tested before. Since standard CBT has been shown to have limited effects in previous outcome studies, it was considered to compare standard CBT with a more interpersonal oriented approach. Although the focus of I-CBT is on more interpersonal issues, it is still the traditional CBT approach of identifying how they think, feel and behave in the situation. Moreover, underlying beliefs are identified and challenged. The treatment was based on a biopsychosocial model  in whichThe control group was a waiting-list group in which the participants waited for 16 weeks after baseline before they were post-scored and then offered 8 weeks of I-CBT outside the study. The ideal would have been a control period up to 1 year without intervention, but this was not possible due to ethical considerations.Supervision/internal validity CBT was delivered by clinical psychologists and one psychiatrist. Supervision in both I-CBT (TCS) and standard CBT (EAF) was given to the therapists as a group once a month, or individually if required. All treatment sessions for standard CBT were audio-recorded to make quality assurance and CBT guidance possible. Adequate use of standard CBT was quality assured by an independent observer using the CTACS quality assuring instrument . All treThe primary outcome measure in this study was the SF-36 version 2 physical function subscale, one of eight subscales , presentAssessments were made at baseline (0 weeks), just after randomization, at mid-therapy (13 weeks), post therapy (20 weeks/post-intervention), 4 weeks after booster (29 weeks), and 1 year after baseline (52 weeks).Assessments were made at baseline (0 weeks), just after randomization, post therapy (12 weeks), 4 weeks after booster (21 weeks/post-intervention), and 1 year after baseline (52 weeks).Assessments were made at baseline (0 weeks), just after randomization, halfway through the waiting period (10 weeks), and at the end of the 16 weeks waiting period (18 weeks/post-intervention).For safety monitoring, we registered adverse events, adverse reaction to trial treatment, and active withdrawal from treatment. Adverse events and adverse reactions were defined as any negative clinical symptom change, disorder, or disease occurring during the study period whether related to the treatment or not. The participant-rated Clinical Global Impression scale was used to assess change in overall health during the trial period  . The ther-squared on 0.1). For a two-sided test with 5% significance level and 80% power, we calculated that we needed 40 patients in each group, 120 in total. Assuming a maximum drop-out level of 25%, we calculated that we needed an additional number of 18 patients in each group, 174 patients in total. A mean change in SF-36 physical function score of 10, 10, and 0 for standard, short and WLC groups, respectively, would increase power to 90%.We calculated sample size assuming a mean change in SF-36 physical function subscale score of 10, 5, and 0 from baseline of, respectively, short I-CBT, standard CBT, and WLC group, a ‘within cell‘ standard deviation (SD) on 15 and a correlation between baseline and post-intervention on 0.3  was defined as an improvement of >10 points for the “mean difference, additional effect relative to baseline at post-intervention” or a mean score in SF-36 physical function of >75 post-intervention , 57. NorFor the Chalder fatigue scale, a MID was not pre-defined, but 0.5 of the SD of these measures at baseline is described in literature , 59. In All patients who were enrolled and randomly allocated to treatment were included in the analysis and analyzed in the groups to which they were randomized (intention to treat/ITT) except the patients excluded for becoming pregnant during the trial (n = 4). Due to repeated measures of outcomes, we used a multilevel linear regression model with random slopes in STATA 11 for Windows . This method uses all available information during follow-up and is less susceptible to bias from missing responses under the assumption of missing at random. Each follow-up time-point was added to the model as a dummy variable . To assess differences between intervention groups and the waiting-list group during follow-up, interaction terms between the group allocation and each registration time-point were included in the model. We also tested a model with only participants with complete registrations of the outcome on all follow-up time points . To assess outcome clustering at the patient level, we estimated an intraclass correlation coefficient (ICC) .The funding source, The Liaison Committee for Education, Research and Innovation in Central Norway, had no role in the study design, data collection, data analysis, data interpretation, or writing of this report/article. All the named authors had access to the anonymised data and commented on drafts during the process leading to the final report. The research group had the final responsibility for the decision to submit the report for publication.The study is approved by the Norwegian Regional Committee for Medical and Health Research Ethics  in Norway, no 4.2008.2586. All patients gave written consent to participate in the study. Patients randomized to the control group were offered 8 weeks of individual I-CBT after the predefined waiting period of 16 weeks was completed. The ideal would be to have a control period up to 1 year after baseline without intervention, but this was not possible due to ethical considerations.p = 0.919, age p = 0.055, fatigue p = 0.718, HADS depression p = 0.474, HADS anxiety p = 0.811 and duration of illness p = 0.087). There were no statistically significant differences at baseline between dropouts and non-dropouts .Of the 626 patients assessed for eligibility, 581 (93%) completed medical and psychological examination, while 45 (7%) patients did not start or complete the assessment. Thereafter 253 (40%) patients were excluded for the following various reasons: Patients with fatigue, but who did not meet the CDC 1994 criteria for CFS numbered 154 (24%). Respectively, 52 (8%) and 40 (6%) patients were excluded because of somatic and psychiatric disorder. Seven patients did not have fatigue and where thus mis-referred. That left 328 (52%) assessed patients who met the CDC 1994 criteria for CFS, and of these 236 (72%) were recruited to the study . One-wayp < 0.05, but no significant difference in years of education p > 0.05 . There were neither no statistically significant differences in physical function SF-36 p = 0.291 scores, HADS depression p = 0.254, HADS anxiety p = 0.254 or fatigue p = 0.521 scores when comparing the groups later at 20 weeks from baseline. Regardless, the reported anxiety and depression symptom levels were low, and neither of the groups scored a mean level of anxiety or depression over a cut-off point of >8/21 in HADS at baseline , lack of motivation for the treatment (n = 2), and long distance to treatment centre (n = 2). In the 16-week CBT group, 16 of 78 (20.5%) participants dropped out during treatment due to fatigue (n = 3), poor economy (n = 2), psychosocial strain (n = 2), long distance to treatment centre (n = 2), lack of motivation for the treatment (n = 4), change of therapist (n = 1), current infection (n = 1), and need of further psychiatric evaluation (n = 1). Poor economy in this context means that the participants could not afford to pay for transport to the clinic and therefore could not attend therapy sessions. All baseline data and outcomes registered were analyzed for all three groups.Twenty (8.5%) participants dropped out just after randomization and had only registered baseline data. Thereafter, 21 (9%) participants dropped out during treatment. There was a variation in drop-out frequency between the study groups. In the 8-week I-CBT group, five of 78 (6%) patients dropped out during treatment due to fatigue (t-tests show that both standard CBT [diff. 0.4 p = 0.35 (95%CI −1.5 to 2.3)] and I-CBT [diff. 3.2 p = 0.056 (95% CI −0.1 to 6.5)] groups revealed an unchanged, positive effect on physical function from post-intervention. Between-groups analyses regarding physical function found no significant differences between the treatment groups (95% CI −10.1 to 2.1 p = 0.20) at the 1-year follow-up , however, the between-groups analyses showed no significant mean score differences for fatigue at the 1-year follow-up (95% CI −2.8 to 2.1 p = 0.8). Eventually, paired t-tests showed that both standard CBT [diff. 0.4 (95% CI −1.5 to 2.3 p = 0.68)] and I-CBT [diff. −1.4 (95% CI −3.0 to 0.2 p = 0.0823)] revealed a sustained reduction in fatigue from post-intervention to 1 year follow up.Participants in both treatment groups reported less fatigue than the control group and exceeded the defined MID on 3.0 points reduction in fatigue post-intervention 20 weeks from baseline (post-intervention). The mean additional effect relative to baseline for fatigue at post- intervention was 5.9  and 4.8  points lower compared to WLC for standard CBT and I-CBT, respectively . I-CBT dAt post-intervention, 50% of the standard CBT group and 40% of the I-CBT group reached a predefined SF-36 physical function score of 75 points or more at post-intervention, compared to only 20% in the WLC group . FurtherFor the standard CBT group, there were statistically significant positive changes in five of the seven remaining subdimensions for the SF-36 post-intervention scores  compared to the control group. The most profound improvements were in the “role physical,” “social function,” and “vitality” subdimensions. There were no statistically significant changes in “general health and role emotional” compared to WLC. Eventually, there were no statistically significant changes in any of the remaining seven SF-36 subdimensions for the I-CBT group compared to the control group .More patients rated themselves as “much better” or “very much better” in overall health measured by the Clinical Global Impression Scale (CGI) after both the standard CBT and I-CBT interventions compared to the control group. At post-intervention, 33 and 26% in the standard CBT vs. I-CBT group, respectively, rated themselves as “much better” or “very much better” in overall health compared to 8% in the WLC group. A minority (<5% in each group) rated themselves as “worse” or “very much worse,” but this did not differ substantially between the three groups at post-intervention . The regwithout a graded or systematic approach. Despite the different approach to activity, a general focus on being active in both treatment groups could explain the positive and sustained effect in physical function for both groups at the 1-year follow-up. Forty-eight percent of the participants had a SF-36 physical function mean score of 75 or more at 1-year follow-up. For fatigue, 35% of the participants were in normal range at 1-year follow-up compared to reference values for the Norwegian population . Furthermore, their experiences did not vary between the groups including the WLC group. In contrast, 33% in the standard CBT group, 26% in the I-CBT group and only 8% in the control group reported to be “much better or very much better” post-intervention. At the 1-year follow-up, as many as 23% in the standard CBT group and 30% in the I-CBT group rated themselves as “much better or very much better” in overall health.Both treatments were well-tolerated. According to the CGI measures, very few rated themselves as ‘worse or ‘very much worse‘ post-intervention  may also be due to either duration of, or differences in content of therapy. However, in this trial, regardless of cause, the patients show a lower dropout rate for I-CBT than standard CBT, which may indicate a better compliance in I-CBT. However, since we did not measure compliance explicit in this study, it is just a hypothesis. Nevertheless, analyses showed no statistically significant differences in baseline characteristics when comparing dropouts to non-dropouts. No serious adverse events or reactions were recorded in any of the groups. This suggests that CBT, if delivered as described in the manuals by similarly qualified and trained clinicians, is a well-tolerated and safe treatment for patients with mild to moderate CFS. The finding that many of our participants reported “minimal change” at post-intervention and at the 1-year follow-up suggests that treatment effects can differ between subgroups. It would be of interest to explore whether special characteristics can be found in patients who benefit from CBT, and thus indicate who should be referred to therapy with a prospect of improvement. This is also thermalized in other studies .Our findings were strengthened by the study design  the relatively high number of participants, the standardized multi-disciplinary assessment by trained physicians, psychologists and physical therapists before inclusion, the use of manual-defined CBT provided by competent clinicians, the thorough blood-test examinations as well as the use of CTACS as a quality-assuring instrument.Our trial also had some limitations: There was a delay in registration in Clinical Trials, because the research group was not aware of the registration requirements at the time. The inclusion of patients started 5 months after registration and approval from the Regional Ethics Committee. We excluded patients who were unable to attend the hospital out-patient clinic, which meant that only out-patients with mild to moderate CFS were included. We ended up with ~13% missing data after 8.5% of the patients dropped out just after randomization and 9% during treatment. The drop-out rate is similar to other CBT studies for CFS . MissingThe findings of this study suggest that both standard CBT and the shorter I-CBT-intervention, individually delivered, are safe therapies which improve physical function in patients with mild to moderate CFS. Standard CBT also cause positive effects on fatigue. The positive effects sustained for at least 1 year. Thus, patients with mild and moderate chronic fatigue syndrome diagnosed according to the CDC and Canadian Consensus Criteria should be offered individual CBT with a prospect of improved physical function and a safe fatigue symptom relief, even though anxiety and depression symptom scores are normal.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Norwegian regional ethicks commity, NTNU/REK midt, Det medisinske fakultet, Postboks 8905, 7491 Trondheim, NORWAY. REK-project number: 4.2008.2586. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MG: calculations, figures and tables, writing and reviewing the text, and corresponding author. EF: planning of the project, writing, and reviewing the text. TS and PB: planning of the project and review of the text. JB: statistics, calculations, figures and tables, and review of the text. All authors contributed to the article and approved the submitted version.TS was the owner of Coperio, a commercial company, from 2005 to May 2022. The study took place at the Pain Clinic, St Olav's University Hospital, Trondheim, Norway. A number of patients received and diagnosed at St Olav's University Hospital had treatment in offices at Coperio, these patients remained patients of St Olav's University Hospital and no patients admitted primarily to Coperio participated in the study. The Coperio Centre has not delivered individual interpersonal and personality-oriented CBT to patients with CFS/ME or other disorders or syndromes either prior to the study or after the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At the end of 2019, a novel coronavirus denominated SARS-CoV-2 rapidly spread through the world causing the pandemic coronavirus disease known as COVID-19. The difference in the inflammatory response against SARS-CoV-2 infection among people living at different altitudes is a variable not yet studied.γ and TNF-α using ELISA assays.A descriptive cross-sectional study was performed in two Peruvian cities at different altitudes for comparison: Lima and Huaraz. Five important proinflammatory cytokines were measured including: IL-6, IL-2, IL-10, IFN-A total of 35 COVID-19 patients and 10 healthy subjects were recruited from each study site. The mean levels of IL-6 (p < 0.03) and TNF-α (p < 0.01) were significantly different among the study groups. In the case of IL-6, patients from Lima had a mean level of 16.2 pg/ml  and 48.3 pg/ml (COVID-19), meanwhile, patients from Huaraz had levels of 67.3 pg/ml  and 97.9 pg/ml (COVID-19). Regarding TNF-α, patients from Lima had a mean level of 25.9 pg/ml  and 61.6 pg/ml (COVID-19), meanwhile, patients from Huaraz had levels of 89.0 pg/ml  and 120.6 pg/ml (COVID-19). The levels of IL-2, IL-10 and IFN-γ were not significantly different in the study groups.Patients with COVID-19 residing at high-altitude tend to have higher levels of inflammatory cytokines compared to patients living at sea level, particularly IL-6 and TNF-α. A better understanding of the inflammatory response in different populations can contribute to the implementation of therapeutic and preventive approaches. Further studies evaluating more patients, a greater variety of cytokines and their clinical impact are required. At the end of 2019, a novel coronavirus denominated SARS-CoV-2 rapidly spread through the world causing the pandemic coronavirus disease known as COVID-19 . CriticaHigh-altitude has been proposed as a protective factor against COVID-19, as lower incidence and fatality rate have been reported in high-altitude areas compared to sea level locations –10. MoreA descriptive cross-sectional study was performed in two Peruvian cities at different altitudes for comparison: Lima and Huaraz. Firstly, Lima, which is the capital of Peru, located in the coastal area and at sea level. Lima has a population of 8,574,974, with most of the people living in urban areas. Also, Huaraz is an Andean city located in the region of Ancash, with an altitude of approximately 3052 m above sea level. Ancash has a population of 1,083,519, with most of the people living in rural areas.Patients hospitalized in healthcare centers and healthy subjects from Lima and Huaraz were enrolled for comparison of cytokines levels. Participants had to be long term residents in their respective city of study, defined as living in the location for at least 1 year. We included patients hospitalized in COVID-19 wards with mild or moderate disease as defined by Peruvian national guidelines. Mild cases included patients presenting with 2 or more of the following symptoms: cough, fever, rhinorrhea, malaise, sore throat and no severe respiratory distress. Moderate cases included patients with 2 or more of the following symptoms: dyspnea, respiratory rate of > 22, oxygen saturation < 95%, chest X-ray compatible with pneumonia, mental state alteration or hypotension.For the control group, healthy subjects from each study site who tested negative for SARS-CoV-2 were recruited. Patients from the disease group and control group were matched by age and gender.Exclusion criteria were patients with previous chronic pulmonary diseases, autoimmune diseases, immunodeficiencies, pregnant women and patients taking immunosuppressant drugs.Instituto de Investigación Nutricional, Lima, Peru (N° 395-2020/CIEI-IIN). The samples were obtained in the context of the epidemiological/syndromic surveillance program according to the health directives of the National Center for Epidemiology, Disease Control and Prevention of the Ministry of Health of Peru. All methods were performed in accordance with the relevant guidelines and regulation. All the subjects signed a written informed consent for the detection of SARS-CoV-2 in nasopharyngeal samples and allowed the use of the samples for other procedures.The study protocol was approved by the Research Ethics Board of the A total of 35 COVID-19 patients and 10 healthy subjects were recruited from each study site. Patients were diagnosed with COVID-19 according to Peruvian national guidelines. Patients were considered with a presumptive diagnosis of COVID-19 if attended with an acute respiratory infection presenting cough, sore throat and at least one of the following symptoms: malaise, fever, headache, dyspnea, rhinorrhea or patients with respiratory distress requiring hospitalization. A RT-PCR test from nasopharyngeal samples was used for the detection of SARS-CoV-2 was carried out on patients with suspected infection.γ and TNF-α. Levels of cytokines were quantified using High Sensitivity Human ELISA Kit corresponding for each cytokine . Each serum sample was run in duplicate, in accordance with the manufacturer’s instructions.Five important proinflammatory cytokines were measured including: IL-6, IL-2, IL-10, IFN-Cytokines levels were calculated and their mean, maximum and minimum level were reported according to each study group. The difference between means was compared using the ANOVA and post-hoc Turkey Test, a value of p < 0.05 was considered significant. Cytokine levels were divided into three quartiles and categorized into 4 ranges. Percentages were calculated according to each quartile of cytokine level and stacked graphs were created using the software GraphPad Prism 9 .γ and TNF-α) and the minimum, maximum and mean of each group is shown in Table γ compared to patients from Lima, regardless of their health status.A total of 35 COVID-19 patients and 10 healthy subjects were recruited from each study site. Cytokine levels were measured , on the contrary, COVID-19 patients show greater percentage in upper values. In the case of TNF-α, a clear tendency can be observed, with more healthy subjects from Lima showing values in the lower quartiles, while most of the healthy and COVID-19 patients from Huaraz are categorized in the upper ranges. A clear pattern in the graphs of IL-2 and IFN-γ could not be observed, as large heterogeneity was observed among groups.COVID-19 is a complex and heterogeneous disease caused by SARS-CoV-2. Its clinical presentation, severity and prognosis are highly variable and depend on several factors. Two of the most important variables with probable clinical impact are the levels pro-inflammatory cytokines levels and the residing altitude of the patients. Currently, there are approximately 400 million people who are permanently living at high altitude, which is considered at elevations higher than 1500 m above sea level . The higHowever, the interplay between high altitude and inflammation has not been addressed in the context of COVID-19, we consider that this three-way interaction may have important clinical implications , 15, 16.Our analysis show that the mean levels of IL-6 and TNF-α were significantly different in the study groups, with higher levels in COVID-19 patients and those residing at high altitude. Levels of IL-2, IL-10 and IFN-γ did not show a significant difference among groups, however, when comparing the study groups, COVID-19 patients in high altitude showed a tendency to present high levels of these cytokines. These findings are in agreement with previous reports of studies evaluating cytokines levels in COVID-19 patients living at sea level. For example, Yang et al.  determinThere are multiple studies reporting the association between different cytokines and COVID-19 disease –23, howeOn the other hand, other factors apart from inflammation may explain the better outcomes in patients at high altitude. Environmental conditions related to barometric pressure, oxygen availability and UV radiation may also have a role in disease severity. Finally, physiologic adaptations in these subjects could explain the better outcomes in these patients as they have a better tolerance to hypoxia . The understanding of these variables could contribute to a better understanding of the pathogenesis of COVID-19 in these populations and consider therapeutic and preventive measures.Our results evidence that patients with COVID-19 residing in high-altitude tend to have higher levels of inflammatory cytokines, particularly IL-6, IL-10 and TNF-α.These findings contribute to the understanding of the response to COVID-19 infection in people residing at different altitudes. Other factors should also be evaluated in these populations, as chronic hypoxia may contribute to different physiological adaptations.A better understanding of the inflammatory response in different populations can contribute to the implementation of therapeutic and preventive approaches. Further studies evaluating more patients, a greater variety of cytokines and their clinical impact are required.Firstly, as this was a first exploratory analysis on the subject, the sample size was limited. Also, a semi quantitative method to measure cytokine level was used, such as ELISA. The association between clinical characteristics and cytokines levels was not evaluated in the current study, given our study design and limitations. Another important limitation is that levels of ACE-2 were not measured, which could have an impact on the expression of inflammatory cytokines. Finally, follow-up was not performed, and it would be interesting to evaluate the changes of cytokines according to day of infection and post-infection.


Additionally, to identify the intracellular targets of exosomal miRNAs in humans, we focused on previously published data, i.e., microarray data and mass spectrometry data in liver samples from ACLF patients. The biological functions and signaling pathways associated with differentially expressed genes were predicted using gene ontology and Kyoto Encyclopedia of Genes and Genomics enrichment analyses; hub genes were also screened based on pathway analysis and the prediction of protein-protein interaction networks. Finally, we constructed the hub gene-miRNA network and performed correlation analysis and qPCR validation. Importantly, our data revealed that MSCs could regulate the miRNA content within exosomes in the hepatic microenvironment. MiR-20a-5p was down-regulated in ACLF hepatocytes and their exosomes, while the levels of chemokine C-X-C Motif Chemokine Ligand 8  were increased in hepatocytes. Importantly, co-culture with hBMSCs resulted in up-regulated expression of miR-20a-5p in exosomes and hepatocytes, and down-regulated expression of CXCL8 in hepatocytes. Altogether, our data suggest that the exosomal miR-20a-5p/intracellular CXCL8 axis may play an important role in the reduction of liver inflammation in ACLF in the context of MSC-based therapies and highlights CXCL8 as a potential target for alleviating liver injury.Acute-on-chronic liver failure (ACLF) is a severe syndrome associated with high mortality. Alterations in the liver microenvironment are one of the vital causes of immune damage and liver dysfunction. Human bone marrow mesenchymal stem cells (hBMSCs) have been reported to alleviate liver injury via exosome-mediated signaling; of note, miRNAs are one of the most important cargoes in exosomes. Importantly, the miRNAs within exosomes in the hepatic microenvironment may mediate the mesenchymal stem cell (MSC)-derived regulation of liver function. This study investigated the hepatocyte exosomal miRNAs which are regulated by MSCs and the target genes which have potential in the treatment of liver failure. Briefly, ACLF was induced in mice using carbon tetrachloride and primary hepatocytes were isolated and co-cultured (or not) with MSCs under serum-free conditions. Exosomes were then collected, and the expression of exosomal miRNAs was assessed using next-generation sequencing; a comparison was performed between liver cells from healthy  Acute-on-chronic liver failure (ACLF) refers to the acute deterioration of liver function in patients with chronic liver disease; it is associated with high mortality rates (from 30% to 50%) among non-transplanted patients worldwide . The mosClinicalTrials.gov. NCT01322906)(Our previous finding (01322906) and the 01322906) suggest 01322906). Additio01322906); howeverIn the liver microenvironment, exosomes play an important role in maintaining the growth of hepatocytes; for instance, primary murine hepatocyte-derived exosomes were shown to promote the proliferation of hepatocytes in a dose-dependent manner . MSCs reversus healthy liver tissues) are enriched in immune response-related genes (up-regulated) in the liver of HBV-ACLF patients (egulated). The altThe use of experimental animals in this study has been approved by the Laboratory Animal Ethics Committee of Guangzhou Forevergen Biosciences (NO.IACUC-G16020). 72 male Balb/c mice  were randomly divided into NC group and ACLF group at a ratio of 1:2, and were fed at specific pathogen-free level. For inducing ACLF, mice were intraperitoneally injected with 10% carbon tetrachloride  twice a week for 8 consecutive weeks, to induce chronic liver fibrosis. Three days later, mice were intraperitoneally injected with 50% carbon tetrachloride (4 ml/kg), resulting in acute liver failure. The control group was injected intraperitoneally with the same dose of olive oil .Histological assessment was performed in the livers of 6 mice in each group to characterize the ACLF model. Briefly, mice were anesthetized intraperitoneally with 4% chloral hydrate  and euthanized 6 h after the last injection with olive oil or carbon tetrachloride. The liver tissues were then dissected and isolated, embedded in paraffin, sectioned and stained; the Sirius red, Masson’s trichrome , and hematoxylin and eosin (H&E) staining techniques were used to evaluate collagen deposition, fibrosis, and inflammation and necrosis, respectively .in vitro model used to reproduce the hepatic microenvironment is schematized in 6 living cells/well in HepatoZYME serum-free media . Identify primary hepatocytes through cell morphology and albumin expression.Western blotting was used to measure the expression of albumin (ab207327).β-actin (ab8227) were used as reference.The 5 living cells/well on a transwell filter  and maintained in Human MSC serum-free culture medium .Human BMSCs were obtained from the Key Laboratory of Stem Cell and Tissue Engineering, Sun Yat-sen University. hBMSCs were characterized using previously reported protocols . Human B2. The supernatants were collected every 48 h ; the collection time-points were 2, 4, 6, 8, and 10 days of culture . Then the cell supernatants were used to isolate exosomes.After 24 h, the culture medium in the three groups of primary murine hepatocytes was removed and fresh culture medium was added (human MSC serum-free medium (SFM) and HepatoZYME-SFM at a 1:1 ratio). In the MSC-treated group, hBMSCs pre-planted on transwell filter were co-cultured with primary hepatocytes in 6-well plates. The culture conditions were 1.5 ml SFM per well, 37°C, and 5% COg for 20 min and cell debris were subsequently removed by centrifugation at 10,000 g for 30 min. An ultrafiltration membrane was then used for concentration . Finally, the exosomes were isolated and purified using the ExO-Quick kit according to the manufacturer’s instructions . The resultant exosomes were then stored at −80°C until further analysis.Exosomes were isolated using the method proposed by Exosomes were identified in accordance with the 2018 guidelines of the International Society for Extracellular Vesicles (ISEV). The ult® Multiplex Small RNA Library kit was used to construct the cDNA library . The conditions of reverse transcription are set strictly according to the manufacturer’s instructions. PCR was performed under the following cycle conditions: 94°C for 30 s, 15 cycles of 94°C for 15 s, 62°C for 30 s, and 70°C for 30 s, followed by 70°C for 5min. Reactions were run on an ABI Step One Plus Real-Time PCR system. PCR products were purified using PAGE. The constructed library was then used for quality and yield detection using an Agilent 2100 Bioanalyzer . Deep sequencing was performed on an Illumina HiSeq 2000 system. The raw data were analyzed using FastQC1. Clean reads were annotated in the miRBase (mature mouse miRNA database) to obtain the known miRNAs. MiRNA quantitation was represented as RPM (reads per million). The RPM is normalized by Z-score transformation using an R-language based online tool called OmicShare2. Differentially expressed exosomal miRNAs were identified according to Z values. The sequencing datas were deposited to the Sequence Read Archive repository3. Accession number is PRJNA689768.In accordance with the requirements for operating the Illumina platform, the methods used in this paper were adapted from 4. The accession numbers used were GSE62030 (miRNA expression), and GSE14668, GSE38941, GSE62029 and GSE96851 (mRNA expression). The DEG article .6 was used; the threshold was set as 0.7.MiRNAs with consistent expression in exosomes and liver tissues were screened. For predicting the target genes of multiple miRNAs, DIANA Micro-CDS v5.0The target genes were defined as those with expression trends opposite to those of miRNAs. The Venn analysis was used to screen the target genes that were up-regulated or down-regulated in the context of transcriptome or proteome data. DEGs with statistical differences in independent studies were included in Venn analysis to ensure that the DEGs obtained in overlapping regions were statistically significant.p ≤ 0.05 was taken to be significant. Additionally, PPI undirected weighted networks were constructed using STRING7, and clustered using Markov Clustering (MCL) with the inflation parameter set to 3. Gene expression values were conducted randomization to exclude the possibility of spurious correlations occurring by chance.The functional annotation of DEGs was performed using Kyoto Encyclopedia of Genes and Genomics (KEGG) analysis and gene ontology (GO) enrichment analysis. Analysis/visualization was performed using the online tool OmicShare. GSE62029 and GSE62030. Statistics and visualization were performed using the online tool OmicShare. The cut-off value of Pearson’s correlation analysis was set more than or equal to 0.6. q ≤ 0.05 was considered as significant differences.The molecular network topology analysis was performed using the software Cytoscape, version 3.6.1. The type of network constructed as a directed random network. The Pearson’s correlation analysis was performed for hub genes and the respective miRNAs in the microarray datasets with the accession numbers 5 living cells/well, and maintained in RPMI-1640 medium containing 10% FBS and 1% penicillin/streptomycin . “Acute liver failure” was induced by treating with 0.1 mM carbon tetrachloride for 12 h  and seeded into 6-well culture plates  at a density of 5 × 10for 12 h . Then, hSense: UUCUCCGAACGUGUCACGUTT; Antisense: ACGUGACACGUU-CGGAGAATT), miR-20a-5p mimic , inhibitor NC (CAGUACUUUUGUGUAGUACAA) and miR-20a-5p inhibitor (CUACCUGCACUAUAAGCACUUUA) were designed and synthesized by GenePharma Company  (Attached ®) and Opti-MEM (Gibco) to transfect siRNA, and perform transfection according to the standard procedures of the manufacturer’s instructions.L02 cells were transfected with miR-20a-5p mimic, mimic NC, miR-20a-5p inhibitor, or inhibitor NC. After 24 h, 0.1 mM carbon tetrachloride was used to induce acute cell injury for 12 h, and the hepatocytes were co-cultured with or without hBMSC for 24 h. Collect RNA. The sequences of mimic NC , and the TB Green Premix EX Tag Kit  were used. Histopathological analysis of the liver of ACLF mice revealed pronounced collagen deposition, fibrosis, inflammatory infiltration, and necrosis . The isoversus the NC group). Moreover, 90 miRNAs were found to be up-regulated and 398 miRNAs were found to be down-regulated in the MSC-treated group relative to that in the ACLF group , leptin receptor (LEPR), C-X-C motif chemokine ligand 2 (CXCL2), interleukin 1 receptor antagonist (IL1RN), interleukin 1 receptor accessory protein (IL1RAP), and 4 were up-regulated CXCL6, CXCL8 , CXCL14, and death receptor 6  : small molecule synthesis, lipid metabolism, drug metabolism, and redox among others . The affNFRSF21) .CXCL8) had a higher Neighborhood Connectivity score. These molecules may serve as important nodes in the network. Last but not least, Pearson’s correlation analysis revealed a moderate to the very strong negative correlation between the expression of miRNAs and that of their target genes in ACLF , formyl peptide receptor 3 (FPR3), somatostatin receptor 1 (SSTR1), adenylate cyclase 1 (ADCY1), guanylate cyclase 1 soluble subunit alpha 2 (GUCY1A2), phosphodiesterase 11A (PDE11A), IL1RN, and IL1RAP . Moreove in ACLF .8 revealed that the 3′-UTR of the CXCL8 transcript matched the seed sequence of miR-20a-5p in its 8-mer form (highest specificity), with a regulatory relationship score of 99 ; its up-regulation suggests short-term high mortality . Therefore of 99 . With rere of 99 . Additioh hBMSCs . In carbh hBMSCs .Based on these results, we speculate that the decrease of miR-20a-5p in the hepatocytes in the context of ACLF leads to an increase in CXCL8 synthesis. To close the model, exosomal miR-20a-5p in the hepatic microenvironment increases after treatment with MSCs, leading to the inhibition of the expression of CXCL8 .In this study, the landscape of exosomal miRNAs from primary hepatocytes of ACLF mice in the presence or absence of MSCs was determined for the first time by high-throughput sequencing. Exosomes are a major method of secretion and transport of extracellular miRNAs . ImportaExisting high-throughput studies in the context of ACLF lack multi-omics combined analyses. In this study, 36 dysregulated miRNAs and 238 DEGs were successfully identified via the analysis of dysregulated exosomal miRNAs and hepatocytes miRNA/mRNA/proteins in the context of ACLF. miRNA-mediated gene expression abnormalities have been identified in immune destabilization, oxidative stress, programmed death, and other processes . Given tThe uncontrolled activation of intrinsic immunity is a prerequisite for the development of systemic inflammation in ACLF. The chemokine cascade is a key step in the recruitment and activation of neutrophils in the liver of ACLF patients . The proThe down-regulation of hepatic miR-20a-5p is associated with the development of inflammation and fibrosis , while tin vitro (data not shown). Therefore, it is quite possible that MSCs deliver miR-20a-5p to hepatocytes through their own exosomes. However, we cannot exclude the possibility that MSC-derived exosomes promote endogenous miR-20a-5p synthesis in hepatocytes through unknown mechanisms. The accurate detection of miR-20a-5p as well as of its precursors should be performed to clarify the above doubt.It would be interesting to investigate whether the up-regulated exosomal miR-20a-5p originated from hBMSCs or hepatocytes. According to published data, miR-20a-5p was detected in hBMSC-derived exosomes, accounting for 0.11% of total exosomal miRNAs . Remarkaversus murine-derived hepatocytes in the context of ACLF. The use of ACLF humanized mice or the direct use of primary hepatocytes obtained from ACLF patients after transplantation would be a better option; however, there are technical and ethical limitations for the time being. The functional analysis of exosomal miRNAs may be important for the understanding of the hepatic pathological mechanisms and/or for the identification of the therapeutic targets  can be found in the article/The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Guangzhou Forevergen Biosciences.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ABCA4-associated) retinopathy. We aimed to quantify ellipsoid zone (EZ) loss and photoreceptor degeneration beyond EZ-loss in ABCA4-associated retinopathy and investigate associations between photoreceptor degeneration, genotype, and age.Outcome measures sensitive to disease progression are needed for ATP-binding cassette, sub-family A, member 4–associated  were segmented using a deep learning (DL) approach. For genotype-phenotype analysis, a model of ABCA4 variants was applied with the age of criterion EZ-loss (6.25 mm2) as the dependent variable.We analyzed 132 eyes from 66 patients (of 67 enrolled) with molecularly confirmed EZ-loss progression rate of [95% CI] 0.09 mm/y . Outer nuclear layer (ONL) thinning extended beyond the area of EZ-loss. The average distance from the EZ-loss boundary to normalization of ONL thickness (to ±2 z score units) was 3.20° . Inner segment (IS) and outer segment (OS) thinning was less pronounced, with an average distance from the EZ-loss boundary to layer thickness normalization of 1.20°  for the IS and 0.60°  for the OS. An additive model of allele severity explained 52.7% of variability in the age of criterion EZ-loss.Patients exhibited an average (square-root-transformed) ABCA4-associated retinopathy exhibited significant alterations of photoreceptors outside of EZ-loss. DL-based analysis of photoreceptor laminae may help monitor disease progression and estimate the severity of ABCA4 variants.Patients with ClinicalTrials.gov identifier: NCT01736293.National Eye Institute Intramural Research Program and German Research Foundation grant PF950/1-1.  ABCA4 gene  coding for transmembrane transporter protein  is the most common cause of inherited macular dystrophy, with a prevalence between 1 in 8000 to 10,000  atrophy, and progression of these areas is being measured by short-wavelength hypoautofluorescence (hypoFAF) , 8. ChanEZ-loss represents a binary metric . As such, information pertaining to more subtle degeneration of photoreceptors beyond the boundaries of EZ-loss is not captured.At the level of an individual A-scan, quantification of EZ-loss is not ideal for evaluating disease severity in the Abca4−/− mouse model due to the limited axial resolution of OCT imaging in mice. Thus, direct comparison of experimental studies in animal models and human data is challenging  across patients, which resulted in the systematic exclusion of patients with progressed disease  ETDRS-grid-based thinning of photoreceptor laminae, and (iii) photoreceptor laminae thinning outside of EZ-loss using a potentially novel, individualized, contour line–based approach. In addition, (iv) this study provides a framework to impute the age of criterion EZ-loss (on the level of each eye) to quantify the severity of individual ABCA4 variants in an interval-scaled manner.The purpose of this study was to quantify and compare photoreceptor degeneration and its change over time in terms of (i) https://doi.org/10.1172/jci.insight.155373DS1).From a total of 67 study participants, 132 eyes from 66 patients were included in this analysis  before or after the study were also available (cf. Methods for details). Since linear mixed models can handle unbalanced repeated measures data, these additional SD-OCT data were also included in the analyses. With these additional visits, the overall median follow-up time was 5.0 years . Throughout this article, the first visit refers to the first visit with imaging data available, while baseline refers to the baseline visit of the prospective natural history study .At baseline, the median [IQR] acuity for the better eyes was 0.8 logMAR   and 0.86 logMAR   for the worse eyes. Best corrected visual acuity worsened slightly over time with a rate of (mixed model estimate [95% CI]) 0.01 logMAR/y [0.01–0.02] .EZ-loss based on the complete absence of photoreceptor outer segments , with little to no bias  pipeline allowed for automated generation of photoreceptor thickness maps and segmentation of segments . Validatsegments . LikewisEZ-loss area over time. The rate of progression was approximately linear after the square-root transformation of the EZ-loss area. Using a “nonparametric profile maximum likelihood” approach, a Box-Cox transformation with λ = 0.4 was optimal to achieve normality of the response distribution in a random-effects model . A square-root transformation corresponds to the linear progression of EZ-loss along the radius of the lesion and is thus an intuitive transformation  3.32 mm  with an annual progression rate of 0.09 mm/y   0.39 mm2/y .The average (square-root transformed) EZ-loss for each patient. The distance from EZ-loss to the contour line, where retinal layer thickness normalized on average , varied markedly between the layers (e layers . The aveEZ-loss (0.43° contour line). Specifically, the inner retina was thinned by (mixed model estimate [95% CI]) –1.54 z score units , the ONL by –3.60 z score units , IS by –3.34 z score units , and OS by –2.11 z score units  . In contrast, the RPE was slightly thickened directly outside of EZ-loss along the 0.43° contour line with +0.61 z score units  (P < 0.001). The average choroidal thickness was within normal limits at 0.00 z score units  (P = 0.992)  . Only 4 EZ-loss boundary at the first visit. All 3 photoreceptor laminae exhibited significant thinning over time in the immediate junctional zone. The thinning rate was greatest at the 0.43° contour line and less at more distant contour lines. Directly outside of the EZ-loss boundary, the rate of change (mixed model estimate [95% CI]) was –0.14 z score units/y  for the ONL thickness, –0.82 z score units/y  for the IS thickness, and –0.59 z score units/y  for the OS thickness. These values correspond in terms of micrometers to a rate of change at the 0.43° contour line of –1.23 μm/y  for the ONL, –1.41 μm/y  for the IS, and –1.67 μm/y  for the OS.z score units per year , P = 0.431), IS exhibited slight but significant change , and OS exhibited no change . z score  and in micrometers , respectively.At more distant contour lines, the change over time was overall lower . At the ETDRS-grid-based analysis revealed approximately linear thinning over time for all segmented retinal layers except for the inner retina in the central ETDRS subfield  was estimated (EZ-loss. This (arbitrary) criterion was selected since square-root-transformed EZ-loss progression rates were linear in this value range  an stimated . Below, ange cf. . This tiABCA4 variant has an independent, additive contribution to the age of criterion EZ-loss, (EZ-loss (y) derived from the model for 31 variants from 43 patients . By comparison for a patient with p.Gly1961Glu and null variants, the model predicts an age of onset of 41.51 years . The model explained  52.7% of the variability in age of criterion EZ-loss . Despite the rather small sample size available for model fitting (23 – 1 patients), the models explained  24.1% of the variability in the age of criterion EZ-loss  classification of variant variability by Cideciyan and coworkers, who used retinal sensitivity data as a readout . Twelve of 43.5% . Similarof 43.5% .ABCA4-associated retinopathy. Integral to this analysis, we developed and validated a potentially novel method that quantified changes in the thickness of the retinal layers with time along contours equidistant to the EZ-loss boundary. Analysis along a contour line closest to the EZ-loss boundary showed clear evidence of disease progression that was not evident from conventional  ETDRS-grid-based analysis. In addition, an EZ-loss-based approach to define a time-invariant measure of disease severity was proposed. This time-invariant estimate of the age of criterion EZ-loss, which reflects age of onset and subsequent progression rate, allowed generating a  interval-scaled classification for the severity of 31 ABCA4 variants.This study provides a detailed analysis of the progression of photoreceptor loss over time using SD-OCT in EZ-loss , or quantitative autofluorescence (STARTT [EudraCT No. 2018-001496-20]) as outcome measures. While DDAF in STGD1 is well established in terms of reproducibility and progression rates , area of on rates , the bouon rates . Thus, tsitivity  and elecsitivity . Moreovediseases , 26. We 65), areaEZ-loss progression rates were readily quantifiable and compatible with prior estimates from smaller retrospective cohorts  allowed us to generate a hypothetical interval-scaled classification for the severity of 31 ABCA4 variants.Overall, our prospective cohort’s  cohorts , 27. Sim cohorts , 29, squgression . Thus, e atrophy , both diEZ-loss with high reliability, as underscored by the strong correlation of the age of criterion EZ-loss predicted separately for the left and right eyes of patients  may affect the results. Second, the additive model is (most likely) a simplification, and interaction effects between variants might result in a more or less severe phenotype than predicted by the simple sum of the allele severities  findings from previous studies of ABCA4-related retinopathy. Approximately half of the ABCA4 patients in an earlier study using wide-field perimetry exhibited abnormal extramacular cone and rod sensitivity that progressed over time (1.1 log/decade and 0.45 log/decade)  . Likewisinopathy  and thatinopathy . PotentiABCA4-associated retinopathy in a large, genetically well-characterized cohort. However, the en face imaging frame was limited (30° × 15°), which led to the inability to evaluate EZ-loss progression in a small subset of patients (ceiling effect). Based on previous data, the en face B-scan density (distance of 120 μm) used here was sufficient for the accurate quantification of photoreceptor thinning (EZ-loss). As noted above, the genotype-phenotype analysis was based on an additive model. Thus, it could not reflect potential interaction effects. In addition, unobserved genetic variation within and outside of the ABCA4 gene may further influence disease severity.Using a DL-based image segmentation pipeline, this study allowed us to quantify photoreceptor degeneration in thinning . Howeverthinning , 41. TheABCA4-associated retinopathy. This approach allowed us to evaluate in a fully automated manner the progression of conventional biomarkers , as well as contour line–based analysis of photoreceptor degeneration over time. In addition, we demonstrated that the age of EZ-loss is dependent on the genotype and provided estimates for 31 variants, including 16 variants, which we believe have not been previously quantitatively analyzed regarding clinical severity .ABCA4 mutation, and patients had to be 12 years or older. Exclusion criteria were evidence of a systemic condition or ocular disease unrelated to ABCA4 mutations that would complicate the analysis of psychophysical, electrophysiological, or imaging data . Lesion size or visual acuity did not constitute inclusion or exclusion criteria in this study.Sixty-seven patients were recruited between October 2012 and September 2018. Study visits included baseline, 6 months, 1 year, and then yearly visits for 5 years. To be included in this study, STGD1 had to be confirmed based on the clinical phenotype, and presence of at least 1 pathogenic Previously acquired normal data over a wide age range  were included to account for normal aging and retinal topography .Patients underwent 30° × 30° fundus autofluorescence imaging , 30° × 30° infrared reflectance (λ 815 nm) imaging, and 30° × 15° SD-OCT imaging  using a Spectralis HRA+OCT (Heidelberg Engineering).One study patient had to be excluded from the presented analysis due to the lack of SD-OCT volume scans . PrebaseEZ-loss was segmented  to account for age and location-specific variation of normal thickness.Finally, the thickness data were standardized in an A-scan-wise manner  was excluded from the analysis given the previously described unique characteristics of the peri-papillary retina in STGD1 .EZ-loss (mm2), as well as retinal layer thicknesses  along evenly spaced contour lines surrounding the EZ-loss boundary , was calculated from the Riemann sum:The length of central discontinuity of the EZ band was measured manually for each SD-OCT B-scan collected. All measurements were made manually, with each B-scan examined at 800% zoom using the 1:1 μm setting. The area of EZ band loss, kX (μm) = the length of central EZ band discontinuity of the kth B-scan and ΔX (μm) = the distance between B-scans. The start of the intact EZ band was sometimes obvious; more frequently, there was an ambiguous region between the obvious absence or presence of the EZ band. To account for this uncertainty, we applied the following rules to determine the edge of the intact EZ band for each B-scan: starting at the peripheral edge of the scan and moving toward the fovea, the start of the discontinuity was defined as the first evidence of loss of the EZ band. Small breaks in the EZ band (<250 μm) were not counted as loss if there was a section of continuous EZ band measuring at least 200 μm, closer to the fovea. This last condition was designed to disregard disruptions due to flecks, which we observed to be generally <225 μm in size and transitory with time, i.e., presence of a fleck at 1 time point did not mean the absence of EZ band at a subsequent time point.where R. Normal distributed data were summarized by their mean and standard deviation; non-normal data were summarized by their median and IQR. A P value less than 0.05 was considered statistically significant.Statistical analyses were performed in the software environment EZ-loss over time with mixed-effects models was identified using the R package boxcoxmix  were applied with eye nested in patients as random effects terms using the R package lme4  a predefined criterion EZ-loss area (6.25 mm2) was imputed for each eye. This estimated age of criterion EZ-loss provided an age-invariant variable that could be compared to the genotype. Specifically, a linear model was fit to the (square-root) transformed EZ-loss progression data from each eye and applied to infer the age at which the eye was expected to have had a square-root-transformed EZ-loss area of 2.5 mm , the EZ-loss progression rate was determined based on the first 2 visits. For the analysis, an additive model (mixed model) of ABCA4 variants was fitted to the data with the age of criterion EZ-loss as a dependent variable analogous to the ADI analysis previously proposed by Cideciyan and coworkers  (n = 43) with exactly 2 ABCA4 variants and a measurable EZ-loss area in at least 2 visits could be included in this analysis  . Only paanalysis .n = 23), with 2 ABCA4 variants that both occurred with other patients in this study, was used to assess the accuracy of the modeling approach through patient-wise leave-one-out cross-validation .A subset of patients (This study adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board of the National Institutes of Health. Written informed consent was obtained from all participants prior to inclusion in the study. No compensation/incentive was offered to the participants.CAC, WMZ, AT, BGJ, and BPB contributed to research design. CAC, LAH, WMZ, and BPB contributed to data acquisition. MP, BGJ, RBH, EU, MPB, HEAS, ASH, and MAC contributed to analysis and interpretation of data. MP, CAC, LAH, WMZ, RBH, EU, BGJ, and BPB contributed to drafting of the manuscript.